The proposal will characterize glucose permeability in amphibian urinary bladder with the aim of developing a simple model for studying glucose transport in renal tubule. Amphibian urinary bladder reabsorbs water and modifies solute composition of the urine; thus it is functionally analogous to the vertebrate kidney. Most of the proposed work will use tissue from the wood frog, a freeze-tolerant frog in which bladder glucose reabsorption is particularly well developed owing to its need to recover filtered sugar. However, the study also will use tissue from freeze-intolerant, common frogs. The working hypothesis is that the glucose reabsorption in the frog bladder is similar to that in the kidney. Testing this hypothesis, the P.I. will determine roles of the brush-border membrane (BBM) and basolateral membrane (BLM) in the overall glucose absorption process. Using Western blot analyses, he will test the hypothesis that the glucose transport is mediated by SGLT1 and SGLT2 in BBM and by GLUT2 in the BLM. He will confirm the identification and localization of transport proteins, by studying inhibition characteristics of trans-epithelial glucose flux using inflated hemibladder preparations in flux chambers, and by studying characteristics of glucose uptake by BBM and BLM vesicles. The hypothesis that glucose transport capacity adapts to changes in substrate availability will be tested by measuring changes in transport protein expression in Western blots, and kinetic properties of glucose uptake in BBM and BLM vesicles prepared from epithelium that has been incubated in varying substrate concentrations. The hypothesis that hypoxia upregulates transporter activity in these membranes will be similarly tested.